title: Novel multiplex bead-based assay for detection of IDH1 and IDH2 mutations in myeloid malignancies.
abstract: Isocitrate dehydrogenase 1 and 2 (IDH) mutations are frequently found in various cancer types such as gliomas, chondrosarcomas and myeloid malignancies. Their molecular detection has recently gained wide recognition in the diagnosis and prognosis of these neoplasms. For that purpose various molecular approaches have been used but a universally accepted method is still lacking. In this study we aimed to develop a novel bead-based liquid assay using Locked nucleic acids (LNA)-modified oligonucleotide probes for multiplexed detection of the most frequent IDH1 (p.R132C, p.R132G, p.R132H, p.R132L, p.R132S) and IDH2 (p.R140Q, p.R172K) mutations. The method includes four steps: 1) PCR amplification of the targeted fragments with biotinylated primers; 2) Direct hybridization to barcoded microbeads with specific LNA-modified oligonucleotide probes; 3) Incubation with phycoerythrin coupled streptavidin; 4) Acquisition of fluorescent intensities of each set of beads on a flow platform (LuminexCorp., USA). We tested the performance of the assay on both artificial plasmid constructs and on clinical samples from 114 patients with known or suspected myeloid malignancies. The method appeared to be superior to direct sequencing having a much higher sensitivity of 2.5% mutant alleles. Applying this method to patients' samples we identified a total of 9 mutations (one IDH1 p.R132C, seven IDH2 p.R140Q and one IDH2 p.R172K). In conclusion, this method could be successfully implemented in the diagnostic work-up for various tumors known to harbor IDH1/2 mutations (e.g. myeloid malignancies, gliomas, etc.). International initiatives are needed to validate the different existing methods for detection of IDH1/2 mutations in clinical settings.
authors.affiliation: Laboratory of Hematopathology and Immunology, National Hematology Hospital, Sofia, Bulgaria.
authors.forename: Velizar
authors.initials: V
authors.lastname: Shivarov
authors.forename: Milena
authors.initials: M
authors.lastname: Ivanova
authors.forename: Evgueniy
authors.initials: E
authors.lastname: Hadjiev
authors.forename: Elissaveta
authors.initials: E
authors.lastname: Naumova
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D005434:Flow Cytometry; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics*; D007951:Leukemia, Myeloid / Q000235:genetics*; D008863:Microspheres; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D010799:Phycoerythrin; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D019809:Streptavidin
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes; D010799:Phycoerythrin; D019809:Streptavidin; C538784:IDH2 protein, human; D007521:Isocitrate Dehydrogenase; C543588:IDH1 protein, human